Project Summary Ribosome biogenesis, a complex cellular process for making the ribosome in the nucleolus, is essential for normal cell growth and proliferation. Defects in ribosome biogenesis are associated with a group of diseases called ribosomopathies. Thus, it is crucial to understand how ribosome biogenesis is properly regulated during normal cell homeostasis. It has been shown that SUMOylation plays a key role in ribosome biogenesis in the nucleolus, including rRNA synthesis and processing, ribosome subunit assembly, maturation and nuclear export. Yet, a SUMO ligase (E3) mediating nucleolar SUMOylation has not been identified. We recently discovered that the deubiquitinating enzyme USP36 is a novel nucleolar SUMO E3. Overexpression of USP36 promotes SUMOylation, mainly in the nucleolus, whereas knockdown of USP36 drastically reduces the levels of SUMOylation in cells. We show that USP36 directly binds to Ubc9 (SUMO E2) and SUMO, and possesses SUMO E3 activity in vitro in reconstituted systems, demonstrating that USP36 is a novel bona fide SUMO E3. The SUMO E3 activity is mapped to an N-terminal region (amino acids 120-300). We further found that USP36 mediates the SUMOylation of Nop58 and Nhp2, components of the box C/D and box H/ACA small nucleolar ribonucleoprotein (snoRNP) complexes, respectively, and regulates their binding to snoRNAs. Together, these data lead to a novel hypothesis that USP36 functions as a crucial SUMO E3 mediating nucleolar SUMOylation and thus being critical for ribosome biogenesis. To gain further insight into the role of USP36 in the regulation of nucleolar SUMOylation and ribosome biogenesis, we will investigate the molecular and biochemical mechanisms underlying the SUMO E3 activity of USP36 in Aim 1, including how the N-terminal SUMO E3 domain contributes to USP36's SUMO E3 activity, how USP36 promotes poly-SUMO chain formation and SUMO transfer to substrates, and how its SUMO E3 and Dub activity are determined. We will then elucidate the nucleolar functions of USP36 as a SUMO E3 in snoRNP and ribosome biogenesis in Aim 2, including whether USP36 regulates snoRNP and ribosome biogenesis-related protein group SUMOylation, whether it regulates rRNA modifications and rRNA processing, and whether it also regulates ribosome subunit maturation and nuclear export. Finally, we will elucidate whether USP36's nucleolar SUMO E3 activity is critical for cell growth and proliferation as well as tissue homeostasis and regeneration in vivo using inducible USP36 knockout mice models in Aim 3. Achieving these goals will provide critical insight into how USP36 properly regulates SUMOylation in the nucleolus and how it regulates ribosome biogenesis.